This application is to explore the function in attachment and biofilm formation of cluster II chemotaxis-like genes found in the opportunistic human pathogen Pseudomonas aeruginosa. P. aeruginosa has multiple sets of chemotaxis-like genes arranged in clusters. Genes in clusters I/V are absolutely required for flagella-mediated chemotaxis. Those in cluster IV are required for pilus-mediated motility and response. Accumulating evidence suggests that some chemotaxis-like genes from bacteria may not be primarily involved in chemotaxis or motility. Such appears to be the case for P. aeruginosa cluster II genes. Cluster II che-like and mcp-like genes are involved in attachment of cells to surfaces and biofilm formation. They appear to play, at most, a minor role in flagella-mediated chemotaxis. Cluster II mutants are not defective in pilus-mediated twitching motility or in swarming across solid surfaces. The long-term objective of the proposed work is to determine the mechanism by which a presumed cluster II signal transduction complex directs cells to attach to surfaces and initiate biofilm formation. In specific aim 1 P. aeruginosa gene chips will be used to identify sets of genes that are transcriptionally activated or repressed by cluster II che-like proteins. Other experiments will explore the possibility that a cluster II signaling complex has a major role in directly modulating the activity of a cell surface molecular machine during the stationary phase of growth. The possibility that cluster II proteins modulate the activities of other proteins through direct physical contact will also be examined. In specific aim 2 genes identified as being regulated by cluster II signal transduction or genes encoding proteins whose activities are modulated by cluster II proteins, will be inactivated by mutation. The attachment and biofilm formation phenotypes of the mutants will be analyzed. Complementary physiological approaches will be used to explore how the activities of the encoded proteins may affect biofilm formation. Cluster II genes are induced in the stationary phase of growth in a cell-density dependant manner. Specific aim 3 will explore the hypothesis that cluster II genes and somemcp genes are coordinately regulated by global regulators that control the exponential-to-stationary phase growth transition in P. aeruginosa. The hypothesis that cluster II proteins interact to form a signal transduction complex will be tested in specific aim 4. Phospho-transfer assays will be carried out and effects of cluster II proteins on the subcellular localization of other green fluorescent tagged cluster II proteins will be determined. Pseudomonas aeruginosa grows as a biofilm in the lungs of cystic fibrosis patients and it grows as a biofilm in infections caused by indwelling medical devices. The proposed work will reveal proteins and genes that are required for biofilm development by prokaryotes.